ABSTRACT
Aim: To evaluate an assay to detect minimum bactericidal concentration (MBC) in Mycobacterium tuberculosis, using as single model rifampicin, isoniazid, levofloxacin (LVX) and linezolid (LNZ) and in combination. Material & methods: MBCs were carried out directly from resazurin microtiter assay plate and 3D checkerboard in M. tuberculosis H37Rv and five resistant clinical isolates. Results: The proposed MBC assay showed similar values to those determined by MGIT™, used as control. LVX and LNZ's MBC values were close to their MIC values. LNZ or LVX combined with isoniazid and rifampicin showed MBC value reduced in 63.7% of the assays. Conclusion: The proposed assay to determine MBCs of drugs can be applied to the study of new compounds with anti-M. tuberculosis activity to detect their bactericidal effect and also in laboratory routine for clinical dose adjustment of drugs according to the patient's profile.
Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Tuberculosis/drug therapy , Drug Resistance, Multiple, Bacterial , Drug Synergism , Humans , Isoniazid/pharmacology , Levofloxacin/pharmacology , Linezolid/pharmacology , Microbial Sensitivity Tests , Rifampin/pharmacologyABSTRACT
BACKGROUND: In recent years, very few effective drugs against Mycobacterium tuberculosis have emerged, which motivates the research with drugs already used in the treatment of tuberculosis. Ethambutol is a bacteriostatic drug that affects cell wall integrity, but the effects of this drug on bacilli are not fully exploited. OBJECTIVE: Based on the need to better investigate the complex mechanism of action of ethambutol, our study presented the proteome profile of M. tuberculosis after different times of ethambutol exposure, aiming to comprehend the dynamics of bacilli response to its effects. M. tuberculosis was exposed to ½ MIC of ethambutol at 24 and 48 hours. The proteins were identified by MALDI-TOF/TOF. RESULTS: The main protein changes occurred in metabolic proteins as dihydrolipoyl dehydrogenase (Rv0462), glutamine synthetase1 (Rv2220), electron transfer flavoprotein subunit beta (Rv3029c) and adenosylhomocysteinase (Rv3248c). CONCLUSION: Considering the functions of these proteins, our results support that the intermediary metabolism and respiration were affected by ethambutol and this disturbance provided proteins that could be explored as additional targets for this drug.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/metabolism , Ethambutol/pharmacology , Mycobacterium tuberculosis/drug effects , Tuberculosis/drug therapy , Antitubercular Agents/therapeutic use , Cell Wall/drug effects , Ethambutol/therapeutic use , Humans , Metabolic Networks and Pathways/drug effects , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/metabolism , Proteome/drug effects , Proteome/isolation & purification , Time Factors , Tuberculosis/microbiologyABSTRACT
AIM: We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. METHODS: The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. RESULTS: EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. CONCLUSION: Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).
Subject(s)
Benzofurans/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Phenols/pharmacology , Proteomics , Bacterial Proteins/drug effects , Bacterial Proteins/metabolism , Benzofurans/chemistry , Citrate (si)-Synthase/drug effects , Electrophoresis, Gel, Two-Dimensional , Genes, Bacterial/drug effects , Humans , Ketol-Acid Reductoisomerase/drug effects , Microbial Sensitivity Tests , Microscopy, Electron, Scanning , Mycobacterium tuberculosis/cytology , Mycobacterium tuberculosis/enzymology , Phenols/chemistry , Phosphoglycerate Kinase/drug effects , Protein Interaction Domains and Motifs , Proteome/analysis , Tandem Mass Spectrometry , Time Factors , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
AIM: To study the proteomic and morphological changes in Mycobacterium tuberculosis H37Rv exposed to subinhibitory concentration of isoniazid (INH). MATERIALS & METHODS: The bacillus was exposed to ½ MIC of INH at 12, 24 and 48 h. The samples' cells were submitted to scanning electron microscopy. The proteins were separated by 2D gel electrophoresis and identified by MS. RESULTS: INH exposure was able to alter the format, the multiplication and causing a cell swelling in the bacillus. The major altered proteins were related to the virulence, detoxification, adaptation, intermediary metabolism and lipid metabolism. CONCLUSION: The protein and morphological changes in M. tuberculosis induced by ½ MIC INH were related to defense mechanism of the bacillus or the action of INH therein.
